Anti-tumour immune responses to modified self-epitopes

ABSTRACT

The present invention relates to modified citrullinated enolase peptides that can be used as targets for cancer immunotherapy. These peptides can be used as vaccines or as targets for monoclonal antibody (mAb) therapy. Such vaccines or mAbs may be used in the treatment of cancer.

The present invention relates generally to modified peptides that can beused as targets for cancer immunotherapy. The modified peptides may becitrullinated enolase peptides. These modified peptides can be used asvaccines or as targets for monoclonal antibody (mAb) therapy. Suchvaccines or mAbs may be used in the treatment of cancer.

In order to be effective, cancer vaccines need to induce a potent immuneresponse which is able to break the tolerance and overcome theimmunosuppressive tumour environment. The importance of CD4 T cells inmediating tumour destruction has been recently highlighted however theinduction of self-specific CD4 responses has proved more difficult. Incontrast, CD4 T cells recognizing modified self-epitopes have been shownto play a role in the pathophysiology of several autoimmune diseasessuch as rheumatoid arthritis (RA), collagen II-induced arthritis,sarcoidosis, celiac disease and psoriasis (Choy, 2012, Grunewald andEklund, 2007, Coimbra et al., 2012, Holmdahl et al., 1985). One of thesecommon modifications is citrullination of arginine which involves theconversion of the positively charge aldimine group (═NH) of arginine tothe neutrally charged ketone group (═O) of citrulline. Citrullination ismediated by Peptidylarginine deiminases (PADs), which are a family ofcalcium dependent enzymes found in a variety of tissues. A recent reportby Ireland et al. 2011, demonstrates that the presentation ofcitrullinated T cell epitopes on APCs is also dependent upon autophagyand PAD activity. This process has also been demonstrated to be anefficient mechanism to enable processing of endogenous antigens forpresentation on MHC class II molecules in professional APCs as well asepithelial cells (Munz, 2012, Schmid et al., 2007). Autophagy isconstitutive in APCs but is only induced by stress in other cells (Greenand Levine, 2014). Thus T cells recognizing citrullinated epitopes haveno target on normal healthy cells. Autophagy is triggered by stress suchas hypoxia and nutrient starvation and is upregulated to promote tumoursurvival (Green and Levine, 2014).

One protein which is citrullinated is enolase, which is a glycolyticenzyme found in the cytoplasm. It is also expressed on the cell surfacewhere it acts as a plasminogen receptor (Miles et al., 1991). In mammalsthere are four isoforms of the enolase enzyme which are encoded by fourdistinct genes (Diaz-Ramos et al., 2012). The ENO1 gene encodes thealpha-enolase which is expressed in almost all adult tissue. ENO2(beta-enolase) is expressed in muscle tissue and ENO3 (gamma-enolase) isfound in neurons (Marangos et al., 1978). More recently a fourth variantENO4 has been identified which is expressed exclusively in sperm(Nakamura et al., 2013). Enzymatically active enolase is formed of twosubunits which can be homo- or heterodimeric (Pancholi, 2001).

ENO1 and ENO3 were identified as citrullinated proteins in the CNS (Janget al., 2008, Jang et al., 2010) and as autoantigen in rheumatoidarthritis (Kinloch et al., 2005, Kinloch et al., 2008, Lundberg et al.,2008, Mandi et al., 2009). More recently monoclonal antibodiesrecognising citrullination of Arg9 in ENO1, Citrullination of Arg9 inENO1 and ENO3 and citrullination of Arg9 in ENO3 have been described(Jang et al., 2012) and have shown that citrullinated enolase lost itsenzymatic activity and was more rapidly degraded by calpain-1 butcitrullination increased ENO1 and ENO3 plasminogen binding activity. Anumber of citrullinated enolase peptides have been described which bindto human MHC II (WO2012138294 A1) and can activate T cells in patientswith rheumatoid arthritis to produce IL-17.

The role of enolase in anaerobic glycolysis and in localisation ofplasminogen on the cell surface have highlighted it as a possible targetfor anti-tumour therapies. Firstly, hypoxia in the tumourmicroenvironment is a major factor during growth of solid tumour.Therefore, upregulation of glycolytic enzymes is necessary for tumoursurvival. The ENO1 promoter contains a hypoxia responsive element whichis upregulated in cells in response to hypoxic stress (Semenza et al.,1996). Secondly, cell surface enolase acts as a plasminogen bindingmolecule allowing the cleavage of plasminogen to plasmin by plasminogenactivator. This process is important for cell invasion with elevatedlevels of plasminogen activator linked to malignancy (Andreasen et al.,2000). Together these functions mean that enolase plays an active rolein tumour growth and metastasis. Multiple approaches have been developedfor targeting enolase therapeutically. ENO1 silencing reducedendometrial cell glycolysis, proliferation and migration in vitro andsignificantly inhibited cell growth in vivo (Zhou et al., 2010).Similarly, administration of anti-ENO1 mAb has been shown to reduce thein vivo growth and metastasis of the human pancreatic cell line CFPAC-1(Principe et al., 2015). ENO1 has been shown to be overexpressed in anumber of cancer tissues including endometrial carcinoma, pancreaticductal adenocarcinoma and non-small cell lung cancer (Zhao et al., 2015,Cappello et al., 2009, Fu et al., 2015). In addition, high enolaseexpression has been correlated with poor clinical outcome for a numberof tumours including breast, hepatocellular and gastric (Reviewed byCapello et al., 2011). In lung cancer, upregulation of ENO1 expressionin tumour cells was observed in 11 out of 17 patients. Higher ENO1expression was correlated with disease recurrence and advanced tumourstage (Chang et al., 2006).

In many patients including pancreatic, leukaemia, melanoma, head andneck, breast and lung ENO1 has been shown to be an autoantigen (Capelloet al., 2011). In pancreatic cancer ENO1 elicits a CD4 and a CD8 T cellresponse both in vitro and in vivo (Cappello et al., 2009) whichinhibited the growth of pancreatic ductal adenocarcinoma cells but nospecific T epitopes were identified. In Head and Neck cancer an HLA-DR8restricted peptide (aa 321-336) stimulated cytotoxic CD4 T cellsresponses (Kondo et al., 2002). In a genetic model of pancreaticadenocarcinoma vaccination with ENO1 DNA elicited humoral and cellularimmune responses against tumours delays tumour progression andsignificantly extended survival (Cappello et al., 2013). The use of wildtype Enolase antigen as a diagnostic and therapeutic agent in cancer hasbeen described (U.S. Pat. No. 7,645,453 B2). In addition, agentstargeting alpha-enolase have been described as a way of increasing thesensitivity of neoplastic cells to chemotherapeutic agents (WO2007072219 A2). We have previously shown that citrullinated Vimentinpeptides can induce CD4-mediated immune responses which result in tumourrejection and long-term survival (WO2014023957 A2). However, all of theimmune responses measured against enolase recognised wild type orphosphorylated enolase but not citrullinated enolase suggesting thatthis enzyme was not citrullinated in cancer.

The inventors have shown that, in normal donors and HLA transgenic mice,there is a repertoire of T cells which recognise citrullinated enolasepeptides and produce IFNγ. They have also shown that certaincitrullinated enolase peptides generate a T cell response in vivo and,as such, can be used as a vaccine target for cancer therapy.

According to a first aspect of the invention, there is provided apeptide comprising, consisting essentially of or consisting of:

-   -   an amino acid sequence selected from:

(Eno1 241-259) VIGMDVAASEFFcitSGKYDLD, (Eno2/3 241-259)VIGMDVAASEFYcitSGKYDLD, (Eno1 21-40) EVDLFTSKGLFcitAAVPSGAS,(Eno3 21-40) EVDLYTAKGLFcitAAVPSGAS, (Eno1 126-145)KGVPLYcitHIADLAGNSEVIL, (mouse Eno1 126-145) KGVPLYcitHIADLAGNPEVIL,(Enol 316-335) VGDDLTVTNPKcitIAKAVNEK or (mouse Eno1 316-335)VGDDLTVTNPKcitIAKAASEK (Eno1 11-25) IFDScitGNPTVEVDLF(Eno3/mouse Eno1 11-25) IFDScitGNPTVEVDLY

wherein “cit” represents citrulline, or

-   -   iii) the amino acid sequence of i), with the exception of 1, 2        or 3 amino acid substitutions, and/or 1, 2 or 3 amino acid        insertions, and/or 1, 2 or 3 amino acid deletions in a        non-citrulline position.

The inventors have unexpectedly found that these citrullinated peptidesderived from enolase can be used to raise an immune response againsttumours including, but not restricted to, pancreatic, leukaemia,melanoma, head and neck, breast and lung tumours. The inventors haveshown that only two peptides

-   -   VIGMDVAASEFFcitSGKYDLD/VIGMDVAASEFYcitSGKYDLD—enolase 241-260        citrullinated at position 253 and    -   EVDLFTSKGLFcitAAVPSGAS/EVDLYTAKGLFcitAAVPSGAS—enolase 21-40        citrullinated at position 32

generated a T cell response in vivo to a citrullinated self enolaseepitope. Three peptides

-   -   KGVPLYcitHIADLAGNSEVIL/KGVPLYcitHIADLAGNPEVIL—enolase 126-145        citrullinated at position 132,    -   VGDDLTVTNPKcitIAKAVNEK/VGDDLTVTNPKcitIAKAASEK—enolase 316-335        citrullinated at position 328 and    -   IFDScitGNPTVEVDLF/IFDScitGNPTVEVDLY—enolase 11-25 citrullinated        at position 15

generated a T cell response in vivo to citrullinated human epitopeswhich were not homologous to mouse and therefore were recognised asforeign antigens.

Citrullinated peptides are known to stimulate T cell responses inautoimmune patients with the shared HLA DR4DR4 motif. In contrast, theinventors are the first to show that enolase 241-260 citrullinated atposition 253 can stimulate potent T cell responses in HLA-DP4 transgenicmice. As HLA-DP4 is expressed by 70% of the population, this makes it apromising vaccine for the treatment of solid tumours. All normal donorsshowing responses to enolase 241-260 citrullinated at position 253expressed HLA-DP4. The response to enolase 241-260 citrullinated atposition 253 was the strongest and showed minimal reactivity to theunmodified sequence. T cells specific for this citrullinated peptideepitope can target tumour cells to elicit strong anti-tumour effects invivo, thus providing the first evidence for the use of citrullinatedenolase 241-260 as a vaccine target for cancer therapy.

ENO1, ENO2 and ENO3 are highly conserved and all express one or theother of the two enolase 241-260 citrullinated at position 253 peptides.In contrast ENO4 has a large inversion at this point in the genesequence. Accordingly, enolase 241-260 citrullinated at position 253, aswell as nucleic acids encoding it, can be used for targeting ENO1, ENO2or ENO3.

Previous studies had shown that enolase 241-260 citrullinated atposition 253 can stimulate T cells responses in RA patients(WO2012138294). As described in detail in the Examples herein, to testwhether the enolase epitopes within a DNA construct were stimulatingimmune responses against citrullinated enolase, mice were immunised withDNA encoding the enolase epitopes and screened for responses against theunmodified and citrullinated epitopes. The mice immunised with DNAencoding the whole enolase sequence responded only to enolase 241-260citrullinated at position 253. This suggests that, when the DNA istranslated, the enolase 241-260 peptide is preferentially citrullinated,the citrullinated peptide binds with higher affinity to MHC or that theT cells stimulated with unmodified enolase epitope recognise thecitrullinated peptides more avidly.

In addition to showing that that encoding epitope within DNA gave acitrullinated T cell responses, enolase 241-260 citrullinated atposition 253 peptide alone stimulated a Th1 responses which onlyrecognised modified epitopes and there was no cross reaction againstwild type peptide. When normal donors were stimulated with enolase241-260 citrullinated at position 253 peptide, 4/6 donors showed aresponse to Enolase 241-260 citrullinated at position 253 but not wildtype peptides. This response was shown to be CD4 Th1 response. In markedcontrast to the RA patients, no IL-17 was produced.

Enolase 241-260 citrullinated at position 253 peptide was immunised witha variety of adjuvants. Immunostimulatory adjuvants such as CpG/MPLA,Poly I:C and Immiquimod stimulated Th1 responses and were required, asthere was no response in the absence of adjuvant. Inert adjuvants suchas Incomplete Freund's adjuvant caused induction of predominantly IL-10responses, suggesting the induction of an iTreg response.

Inserted amino acids and replacement amino acids may be naturallyoccurring amino acids or may be non-naturally occurring amino acids and,for example, may contain a non-natural side chain. If more than oneamino acid residue is substituted and/or inserted, thereplacement/inserted amino acid residues may be the same as each otheror different from one another. Each replacement amino acid may have adifferent side chain to the amino acid being replaced.

Enolase is highly conserved between those species in which the gene hasbeen cloned (chicken, mouse, dog, sheep, cow, horse, pig and human).Accordingly, a peptide of the invention, optionally in combination witha nucleic acid comprising a sequence that encodes such a peptide it, canbe used for treating cancer in non-human mammals.

The invention also includes within its scope peptides having the aminoacid sequence as set out above and sequences having substantial identitythereto, for example, 70%, 80%, 85%, 90%, 95% or 99% identity thereto,as well as their use in medicine and in particular in a method fortreating cancer. The percent identity of two amino acid sequences or oftwo nucleic acid sequences is generally determined by aligning thesequences for optimal comparison purposes (e.g., gaps can be introducedin the first sequence for best alignment with the second sequence) andcomparing the amino acid residues or nucleotides at correspondingpositions. The “best alignment” is an alignment of two sequences thatresults in the highest percent identity. The percent identity isdetermined by comparing the number of identical amino acid residues ornucleotides within the sequences (i.e., % identity=number of identicalpositions/total number of positions×100).

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm known to those of skill inthe art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul modified as in (Karlinand Altschul, 1993). (Karlin and Altschul, 1993). The NBLAST and XBLASTprograms of Altschul, et al. have incorporated such an algorithm(Altschul et al., 1990). BLAST nucleotide searches can be performed withthe NBLAST program, score=100, wordlength=12 to obtain nucleotidesequences homologous to a nucleic acid molecules of the invention. BLASTprotein searches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to a proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in (Altschul et al.,1997). Alternatively, PSI-Blast can be used to perform an iteratedsearch that detects distant relationships between molecules. Whenutilizing BLAST, Gapped BLAST, and PSI-Blast programs, the defaultparameters of the respective programs (e.g., XBLAST and NBLAST) can beused. See http://www.ncbi.nlm.nih.gov. Another example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller (Myers and Miller, 1989). The ALIGN program (version2.0) which is part of the GCG sequence alignment software package hasincorporated such an algorithm. Other algorithms for sequence analysisknown in the art include ADVANCE and ADAM as described in (Torelli andRobotti, 1994) and FASTA described in (Pearson and Lipman, 1988). WithinFASTA, ktup is a control option that sets the sensitivity and speed ofthe search.

Peptides of the invention may be synthesised using Fmoc chemistry orother standard techniques known to those skilled in the art.

Another convenient way of producing a peptide according to the presentinvention is to express the nucleic acid encoding it, by use of nucleicacid in an expression system.

The inventors have shown that that immunisation with nucleic acidsencoding the peptides of the invention gives immune responses tocitrullinated peptides but not to wild type non-citrullinated peptides.Accordingly, the present invention further provides an isolated nucleicacid encoding a peptide of the present invention. In a preferred aspect,the present invention provides a nucleic acid which codes for a peptideof the invention as defined above.

The inventors have also found that administration of nucleic acid, suchas DNA or RNA, encoding full length enolase gives rise to strong immuneresponses to only enolase 241-260 citrullinated at position 253. Thisforms a further aspect of the invention. Examples of the amino acidsequence of full length enolase are provided in the Examples anddrawings herein and it is within the skill of a person skilled in theart to provide nucleic acid sequences that encode such amino acidsequences.

The skilled person will be able to determine substitutions, deletionsand/or additions to such nucleic acids which will still provide apeptide of the present invention. The nucleic acid may be DNA, cDNA, orRNA such as mRNA obtained by cloning or produced by chemical synthesis.For therapeutic use, the nucleic acid is preferably in a form capable ofbeing expressed in the subject to be treated. The peptide of the presentinvention or the nucleic acid of the present invention may be providedas an isolate, in isolated and/or purified form, or free orsubstantially free of material with which it is naturally associated. Inthe case of a nucleic acid, it may be free or substantially free ofnucleic acid flanking the gene in the human genome, except possibly oneor more regulatory sequence(s) for expression. Nucleic acid may bewholly or partially synthetic and may include genomic DNA, cDNA or RNA.Where nucleic acid according to the invention includes RNA, reference tothe sequence shown should be construed as reference to the RNAequivalent, with U substituted for T.

Nucleic acid sequences encoding a peptide of the present invention canbe readily prepared by the skilled person, for example using theinformation and references contained herein and techniques known in theart (for example, see (Sambrook, 1989, Ausubel, 1992)), given thenucleic acid sequences and clones available. These techniques include(i) the use of the polymerase chain reaction (PCR) to amplify samples ofsuch nucleic acid, e.g. from genomic sources, (ii) chemical synthesis,or (iii) preparing cDNA sequences. DNA encoding the polypeptide may begenerated and used in any suitable way known to those of skill in theart, including by taking encoding DNA, identifying suitable restrictionenzyme recognition sites either side of the portion to be expressed, andcutting out said portion from the DNA. The portion may then be operablylinked to a suitable promoter in a standard commercially-availableexpression system. Another recombinant approach is to amplify therelevant portion of the DNA with suitable PCR primers. Modifications tothe sequences can be made, e.g. using site directed mutagenesis, to leadto the expression of modified peptide or to take account of codonpreferences in the host cells used to express the nucleic acid.

The present invention also provides constructs in the form of plasmids,vectors, transcription or expression cassettes which comprise at leastone nucleic acid as described above. The present invention also providesa recombinant host cell which comprises one or more constructs as above.As mentioned, a nucleic acid encoding a peptide of the invention formsan aspect of the present invention, as does a method of production ofthe composition which method comprises expression from encoding nucleicacid. Expression may conveniently be achieved by culturing underappropriate conditions recombinant host cells containing the nucleicacid. Following production by expression, a composition may be isolatedand/or purified using any suitable technique, then used as appropriate.

Systems for cloning and expression of a polypeptide in a variety ofdifferent host cells are well known. Suitable host cells includebacteria, mammalian cells, yeast and baculovirus systems. Mammalian celllines available in the art for expression of a heterologous polypeptideinclude Chinese hamster ovary cells, HeLa cells, baby hamster kidneycells, NSO mouse melanoma cells and many others. A common, preferredbacterial host is E. coli. The expression of antibodies and antibodyfragments in prokaryotic cells such as E. coli is well established inthe art. Expression in eukaryotic cells in culture is also available tothose skilled in the art as an option for production of a specificbinding member, see for recent review, for example (Reff, 1993, Trill etal., 1995). For a review, see for example (Pluckthun, 1991). Expressionin eukaryotic cells in culture is also available to those skilled in theart as an option for production of a specific binding member, see forrecent review, for example (Reff, 1993, Trill et al., 1995).

Suitable vectors can be chosen or constructed, containing appropriateregulatory sequences, including promoter sequences, terminatorsequences, polyadenylation sequences, enhancer sequences, marker genesand other sequences as appropriate. Vectors may be plasmids, viral e.g.‘phage, or phagemid, as appropriate. For further details see, forexample, Molecular Cloning: A Laboratory Manual (Sambrook, 1989). Manyknown techniques and protocols for manipulation of nucleic acid, forexample in preparation of nucleic acid constructs, mutagenesis,sequencing, introduction of DNA into cells and gene expression, andanalysis of proteins, are described in detail in Short Protocols inMolecular Biology (Ausubel, 1992).

Thus, a further aspect of the present invention provides a host cell,which may be isolated, containing nucleic acid as disclosed herein. Astill further aspect provides a method comprising introducing suchnucleic acid into a host cell. The introduction may employ any availabletechnique. For eukaryotic cells, suitable techniques may include calciumphosphate transfection, DEAE-Dextran, electroporation, liposome-mediatedtransfection and transduction using retrovirus or other virus, e.g.vaccinia or, for insect cells, baculovirus. For bacterial cells,suitable techniques may include calcium chloride transformation,electroporation and transfection using bacteriophage. The introductionmay be followed by causing or allowing expression from the nucleic acid,e.g. by culturing host cells under conditions for expression of thegene.

In one embodiment, the nucleic acid of the invention is integrated intothe genome (e.g. chromosome) of the host cell. Integration may bepromoted by inclusion of sequences which promote recombination with thegenome, in accordance with standard techniques.

The present invention also provides a method which comprises using aconstruct as stated above in an expression system in order to express apolypeptide as described above.

Polypeptides of the invention can be used to identify and/or isolatebinding moieties that bind specifically to the polypeptide of theinvention. Such binding moieties may be used as immunotherapeuticreagents and may include antibodies. Therefore, in a further aspect, theinvention provides a binding moiety that binds the polypeptide of theinvention.

The binding moiety of the invention may be an antibody. The term“antibody” as used herein refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site that specifically bindsan antigen, whether natural or partly or wholly synthetically produced.The term “antibody” includes antibody fragments, derivatives, functionalequivalents and homologues of antibodies, humanised antibodies,including any polypeptide comprising an immunoglobulin binding domain,whether natural or wholly or partially synthetic and any polypeptide orprotein having a binding domain which is, or is homologous to, anantibody binding domain. Chimeric molecules comprising an immunoglobulinbinding domain, or equivalent, fused to another polypeptide aretherefore included. Cloning and expression of chimeric antibodies aredescribed in EP-A-0120694 and EP-A-0125023. A humanised antibody may bea modified antibody having the variable regions of a non-human, e.g.murine, antibody and the constant region of a human antibody. Methodsfor making humanised antibodies are described in, for example, U.S. Pat.No. 5,225,539. Examples of antibodies are the immunoglobulin isotypes(e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses;fragments which comprise an antigen binding domain such as Fab, scFv,Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal.A monoclonal antibody may be referred to herein as “mab”.

It is possible to take an antibody, for example a monoclonal antibody,and use recombinant DNA technology to produce other antibodies orchimeric molecules which retain the specificity of the originalantibody. Such techniques may involve introducing DNA encoding theimmunoglobulin variable region, or the complementary determining regions(CDRs), of an antibody to the constant regions, or constant regions plusframework regions, of a different immunoglobulin (see, for instance,EP-A-184187, GB 2188638A or EP-A-239400). A hybridoma (or other cellthat produces antibodies) may be subject to genetic mutation or otherchanges, which may or may not alter the binding specificity ofantibodies produced.

It has been shown that fragments of a whole antibody can perform thefunction of binding antigens. Examples of binding fragments are (i) theFab fragment consisting of VL, VH, CL and CH1 domains; (ii) the Fdfragment consisting of the VH and CH1 domains; (iii) the Fv fragmentconsisting of the VL and VH domains of a single antibody; (iv) the dAbfragment (Ward, E. S. et al., Nature 341:544-546 (1989)) which consistsof a VH domain; (v) isolated CDR regions; (vi) F(ab′)2 fragments, abivalent fragment comprising two linked Fab fragments (vii) single chainFv molecules (scFv), wherein a VH domain and a VL domain are linked by apeptide linker which allows the two domains to associate to form anantigen binding site (Bird et al., Science 242:423-426 (1988); Huston etal., PNAS USA 85:5879-5883 (1988)); (viii) bispecific single chain Fvdimers (PCT/US92/09965) and (ix) “diabodies”, multivalent ormultispecific fragments constructed by gene fusion (WO94/13804; P.Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993)).Diabodies are multimers of polypeptides, each polypeptide comprising afirst domain comprising a binding region of an immunoglobulin lightchain and a second domain comprising a binding region of animmunoglobulin heavy chain, the two domains being linked (e.g. by apeptide linker) but unable to associate with each other to form anantigen binding site: antigen binding sites are formed by theassociation of the first domain of one polypeptide within the multimerwith the second domain of another polypeptide within the multimer(WO94/13804). Where bispecific antibodies are to be used, these may beconventional bispecific antibodies, which can be manufactured in avariety of ways (Hollinger & Winter, Current Opinion Biotechnol.4:446-449 (1993)), e.g. prepared chemically or from hybrid hybridomas,or may be any of the bispecific antibody fragments mentioned above. Itmay be preferable to use scFv dimers or diabodies rather than wholeantibodies. Diabodies and scFv can be constructed without an Fc region,using only variable domains, potentially reducing the effects ofanti-idiotypic reaction. Other forms of bispecific antibodies includethe single chain “Janusins” described in Traunecker et al., EMBO Journal10:3655-3659 (1991). Bispecific diabodies, as opposed to bispecificwhole antibodies, may also be useful because they can be readilyconstructed and expressed in E. coli. Diabodies (and many otherpolypeptides such as antibody fragments) of appropriate bindingspecificities can be readily selected using phage display (WO94/13804)from libraries. If one arm of the diabody is to be kept constant, forinstance, with a specificity directed against antigen X, then a librarycan be made where the other arm is varied and an antibody of appropriatespecificity selected. An “antigen binding domain” is the part of anantibody which comprises the area which specifically binds to and iscomplementary to part or all of an antigen. Where an antigen is large,an antibody may only bind to a particular part of the antigen, whichpart is termed an epitope. An antigen binding domain may be provided byone or more antibody variable domains. An antigen binding domain maycomprise an antibody light chain variable region (VL) and an antibodyheavy chain variable region (VH).

Also encompassed within the present invention are binding moieties basedon engineered protein scaffolds. Protein scaffolds are derived fromstable, soluble, natural protein structures which have been modified toprovide a binding site for a target molecule of interest. Examples ofengineered protein scaffolds include, but are not limited to,affibodies, which are based on the Z-domain of staphylococcal protein Athat provides a binding interface on two of its a-helices (Nygren, P. A.(2008). FEBS J 275(11): 2668-76); anticalins, derived from lipocalins,that incorporate binding sites for small ligands at the open end of abeta-barrel fold (Skerra, A. (2008) FEBS J 275(11): 2677-83),nanobodies, and DARPins. Engineered protein scaffolds are typicallytargeted to bind the same antigenic proteins as antibodies, and arepotential therapeutic agents. They may act as inhibitors or antagonists,or as delivery vehicles to target molecules, such as toxins, to aspecific tissue in vivo (Gebauer, M. and A. Skerra (2009). Curr OpinChem Biol 13(3): 245-55). Short peptides may also be used to bind atarget protein. Phylomers are natural structured peptides derived frombacterial genomes. Such peptides represent a diverse array of proteinstructural folds and can be used to inhibit/disrupt protein-proteininteractions in vivo (Watt, P. M. (2006). Nat Biotechnol 24(2):177-83)].

In a further aspect, the present invention provides a peptide of thefirst aspect and/or a nucleic acid comprising a sequence that encodessuch a peptide and/or a binding moiety of the invention, for use inmedicine.

The invention also provides a peptide of the first aspect and/or anucleic acid comprising a sequence that encodes such a peptide and/or abinding moiety of the invention, for use in a method for treatingcancer, as well as the use of such a peptide and/or nucleic acid and/orbinding moiety, in the manufacture of a medicament for the treatment ofcancer. The invention also provides a method of treating cancer,comprising administering a peptide of the first aspect and/or a nucleicacid comprising a sequence that encodes such a peptide and/or bindingmoiety of the invention to a subject in need of such treatment. Thecancer may be breast cancer including oestrogen receptor negative breastcancer, colorectal cancer, gastric cancer, non-small cell lung cancer,ovarian cancer including endometrial carcinoma, pancreatic cancerincluding pancreatic ductal adenocarcinoma, leukaemia, melanoma, headand neck cancer or lung cancer.

The peptide may be a T or B cell epitope. Peptides in accordance withthe present invention may be used alone or in combination. In addition,they may be used in combination with other therapeutic agents, such asanti-cancer agents including but not limited to checkpoint blockadedrugs such as ipilimumab.

Peptides in accordance with the invention may be delivered in vivo as apeptide, optionally in the form of a peptide as disclosed inWO02/058728. The inventors have surprisingly found that peptides of theinvention give rise to strong immune responses when administered as apeptide. Such peptides may be administered as just the sequence of thepeptide, or as a polypeptide containing the peptide, or even as the fulllength protein. Alternatively, peptide in accordance with the inventionmay be administered in vivo as a nucleic acid encoding the peptide,encoding a polypeptide containing the peptide or even encoding the fulllength protein. Such nucleic acids may be in the form of a mini gene,i.e. encoding a leader sequence and the peptide or a leader sequence andfull length protein. Nucleic acids encoding epitopes useful in thepresent invention may be targeted to antigen presenting cells and othercells that express PAD enzymes, preferably PAD4 enzymes. Nucleic acidsof the present invention may be targeted by including a nucleic acidencoding a targeting agent, such as Fc or a monoclonal antibodytargeting a different antigen on APCs, e.g. anti-DEC205 mAb or by meansof intradermal injection as skin has a large number of APCs.

As used herein, the term “treatment” includes any regime that canbenefit a human or non-human animal. The polypeptide and/or nucleic acidand/or binding moiety may be employed in combination with apharmaceutically acceptable carrier or carriers to form a pharmaceuticalcomposition. Such carriers may include, but are not limited to, saline,buffered saline, dextrose, liposomes, water, glycerol, ethanol andcombinations thereof.

It is envisaged that injections will be the primary route fortherapeutic administration of the compositions of the invention althoughdelivery through a catheter or other surgical tubing may also be used.Some suitable routes of administration include intravenous,subcutaneous, intradermal, intraperitoneal and intramuscularadministration. Liquid formulations may be utilised after reconstitutionfrom powder formulations.

For intravenous injection, or injection at the site of affliction, theactive ingredient will be in the form of a parentally acceptable aqueoussolution which is pyrogen-free, has suitable pH, is isotonic andmaintains stability. Those of relevant skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such assodium chloride injection, Ringer's Injection or Lactated Ringer'sInjection. Preservatives, stabilisers, buffers, antioxidants and/orother additives may be included, as required.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carriersuch as gelatin or an adjuvant. Liquid pharmaceutical compositionsgenerally comprise a liquid carrier such as water, petroleum, animal orvegetable oils, mineral oil or synthetic oil. Physiological salinesolution, dextrose or other saccharide solution or glycols such asethylene glycol, propylene glycol or polyethylene glycol may beincluded. Where the formulation is a liquid it may be, for example, aphysiologic salt solution containing non-phosphate buffer at pH 6.8-7.6,or a lyophilised powder.

The composition may be administered in a localised manner to a tumoursite or other desired site or may be delivered in a manner in which ittargets tumour or other cells.

The compositions are preferably administered to an individual in a“therapeutically effective amount”, this being sufficient to showbenefit to the individual. The actual amount administered, and rate andtime-course of administration, will depend on the nature and severity ofwhat is being treated. Prescription of treatment, e.g. decisions ondosage etc, is within the responsibility of general practitioners andother medical doctors, and typically takes account of the disorder to betreated, the condition of the individual patient, the site of delivery,the method of administration and other factors known to practitioners.The compositions of the invention are particularly relevant to thetreatment of cancer, and in the prevention of the recurrence of suchconditions after initial treatment or surgery. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences (Remington, 1980). A composition may beadministered alone or in combination with other treatments, eithersimultaneously or sequentially dependent upon the condition to betreated. Other cancer treatments include other monoclonal antibodies,other chemotherapeutic agents, other radiotherapy techniques or otherimmunotherapy known in the art. One particular application of thecompositions of the invention is as an adjunct to surgery, i.e. to helpto reduce the risk of cancer reoccurring after a tumour is removed. Thecompositions of the present invention may be generated wholly or partlyby chemical synthesis. The composition can be readily prepared accordingto well-established, standard liquid or, preferably, solid-phase peptidesynthesis methods, general descriptions of which are broadly available(see, for example, in Solid Phase Peptide Synthesis, 2^(nd) edition(Stewart, 1984), in The Practice of Peptide Synthesis (Bodanzsky, 1984)and Applied Biosystems 430A User's Manual, ABI Inc., or they may beprepared in solution, by the liquid phase method or by any combinationof solid-phase, liquid phase and solution chemistry, e.g. by firstcompleting the respective peptide portion and then, if desired andappropriate, after removal of any protecting groups being present, byintroduction of the residue X by reaction of the respective carbonic orsulfonic acid or a reactive derivative thereof.

The polypeptides, complexes, nucleic acid molecules, vectors, cells andbinding moieties of the invention may be non-naturally occurring and/orpurified and/or engineered and/or recombinant and/or isolated and/orsynthetic.

It is preferred of the peptide of the invention does not comprise,consist essentially of or consist of a sequence selected from:

VAASEFFRSGKYDLDFKSPD VAASEFYRSGKYDLDFKSPD TSKGLFcitAAVPSGASTGIYETAKGLcitAAVPSGASTGIYE AGAVEKGVPLYcitHIADLAGN TVTNPKcitIAKAVNEKSCNCL orTVTNPKcitIAKAASEKSCNCL.

Preferred features of each aspect of the invention are as for each ofthe other aspects mutatis mutandis. The prior art documents mentionedherein are incorporated to the fullest extent permitted by law.

EXAMPLES

The present invention will now be described further with reference tothe following examples and the accompanying drawings.

FIG. 1: Sequence Alignments of Human Enolase.

Alignment of Human Enolase Subunits ENOA (ENO1, α), ENOB (ENO3, β), ENOG(ENO2, γ) and ENO4 depicting homology. Light grey homologous regions,dark grey not homologous with ENO4.

FIG. 2. Screening IFNγ Responses to Peptide Pools

Transgenic mouse strains with human DR4 (A) or DR1/HHD (B) and parentalC57BL/6 (C) mice were used to screen IFNγ responses to peptide. Micewere immunised with pools of 4-6 non-overlapping Human citrullinatedEnolase peptides. 14 days after immunisation, mice were sacrificed andsplenocytes were harvests. Ex vivo responses to stimulation with Humanand mouse equivalent peptides were assessed by IFNγ Elispot. Media onlyresponses were used as a negative control. For each pool n=3.Statistical significance of peptide responses compared to mediaresponses for each pool was determined by ANOVA with Dunnett's post-hoctest *p<0.5, **p<0.01, ***p<0.001.

FIG. 3. Human Enolase 241-260 citrullinated Peptide Induces Strong IFNγResponses

A single immunisation of human 241cit peptide was given to transgenicDR4 mice. Ex vivo Elispot was used to determine the IFNγ (A) and IL-10(B) responses generated to the human and mouse equivalent citrullinated(cit) peptides and the wild type (wt) sequences. IFNγ responses tocitrullinated peptides in the presence of MHC class II blocking antibody(L243) were also assessed by Elispot (C). For all assays media onlyresponses were used as a negative control. For each experiment n=3. pvalues are shown.

FIG. 4. Multiple Citrullinated Enolase Peptides Induce Low Level IFNγResponses

Mice were given a single immunisation of citrullinated human Enolasepeptides corresponding to positions 21-40 (A), 126-145 (B) and 316-335(C). Ex vivo Elispot was used to determine IFNγ responses in DR4 andDR1/HHD mice. Responses are shown to human and mouse citrullinatedpeptides and their wild type equivalents when available. Media onlyresponses were used as a negative control. For each peptide n=3.Statistical significance of responses compared to media control weredetermined by ANOVA with Dunnett's post-hoc test*p<0.5, **p<0.01,***p<0.001.

FIG. 5. Enolase 241cit Peptide Induces Responses in DP4 Mice.

Transgenic DP4 mice were immunised with three doses of Enolase 241citpeptide over three weeks. Splenocytes were collected 21 days after theinitial dose was administered. Ex vivo IFNy Elispot were used todetermine the response to Enolase 241 peptides.

FIG. 6. Human Enolase 241cit Peptide Provides an In Vivo SurvivalAdvantage in Anti-Tumour Studies

Immunoblot (A) of lysates from B16F1 (Lane 1), ID8 (Lane 2), TrampC1(Lane 3), Pan02 (Lane 4), LLC/2 (Lane 5), RTLCL (Lane 6), HeLa (Lane 7)cell lines against ladder probed for a Enolase (ENO1) and β actin. Thebands correspond to the expected size for ENO-1 (47 kDa) and β-actin (42kDa).

DR4 mice were challenged with B16DR4 tumour. Survival (B) and tumoursize at day 17 post tumour implant (C) are shown for unimmunised controlanimals and animals immunised with Enolase 241cit peptide four daysafter tumour implant. n=10, results shown are from two independentexperiments.

DR4 mice were challenged with B16 tumour expressing IFNγ inducible DR4.Survival (D) and tumour size at day 17 post tumour implant (E) are shownfor unimmunised control animals and animals immunised with Enolase241cit peptide four days after tumour implant. n=10. Surviving mice fromthe immunised group were rechallenged with the same tumour cell line atday 42 post initial tumour implant (indicated by arrow). Survival datafor rechallenged mice and a previous unchallenged control group areshown (F). Significant p values are shown. For tumour volume medians andp values are shown as determined by Mann Whitney U test.

FIG. 7. Enolase 241cit Peptide Provides a Survival Advantage in an LLC2Tumour Model

DR4 mice were challenged with the Lewis lung carcinoma cell line LLC2.Four days after tumour challenge mice were immunised with human Enolase241cit peptide. Survival data for mice challenged with wild type LLC2(A) or LLC2 transfected with constitutive chimeric DR4 (B) are shown.Statistical differences between immunised and unimmunised control micewere determined by Mantel-Cox test, p values are shown, n=10.

FIG. 8. ENO1 DNA Vaccination Induces a Similar Response to Enolase241cit Peptide

DR4 mice were given two immunisations of ENO1 DNA bullets using a genegun. After 14 days after the second immunisation mice were sacrificedand splenocytes were harvested. Ex vivo Elispots were performed todetermine IFNγ (A) and IL-10 (B) responses to stimulation with Enolase241 peptides. Mice were challenged with B16DR4 tumour cell line whichconstitutively expressed DR4 and after 4 days were immunised with ENO1DNA, survival data (C) and tumour volume at day 11 (D) are shown.

FIG. 9. Adjuvant Effects the Response Induced to Enolase 241cit Peptide.

DR4 mice were given a single immunisation of human Enolase 241cit in thepresence of adjuvant CpG/MPLA or IFA. IFNγ (A) and IL-10 (B) responseswere determined by ex vivo Elispot. For these studies n=3. IFNγresponses in mice given a single or three immunisation of 5 μg or 25 μgof Enolase 241cit peptide in the presence of GM-CSF were determined (C).For these studies n=3. DR4 mice were also challenge with B16DR4 tumourand survival (D) of unimmunised control animals or animals immunisedwith 3 doses of 5 μg of Enolase 241cit peptide with GM-CSF beginningfour days after tumour implant were determined. n=10.

FIG. 10. Responses Develop Rapidly Suggesting a Pre-Existing Enolase241cit Response.

DR4 mice were immunised with a single dose of Enolase 241cit peptide inCpG/MPLA 2, 6 or 14 days before mice were sacrificed and ex vivoElispots were used to determine the IFNγ responses. n=3, p valuesrepresent significant difference compared to peptide responses at day 2.

FIG. 11. Enolase 241cit Peptide Induces Responses in Human PBMCs.

PBMCs were isolated from 6 healthy donors and cultured with media, humanEnolase 241cit or Enolase 241 wt peptide. Thymidine assays wereperformed to determine proliferation after 4, 7 and 11 days (A). HLAtyping was performed on each donor. Supernants from donor 4 on day 11were collected and analysed for cytokine levels using luminex (B). Datashown represents response above media control background for eachcytokine. PBMCs from donor 4 were labelled with CFSE prior tostimulation with wild type and citrullinated peptides. The CD4 and CD8populations within the CFSE labelled cell population was assessed forthe peptide stimulated samples by flow cytometry at day 7 and day 10(C).

FIG. 12.

Alignment of Human Enolase

(ENOA) subunit with equivalent sequences from other species (Mouse, Rat,Cow, Pig, Horse, Chicken, Cat, Dog, Rabbit and Sheep) depictinghomology.

FIG. 13.

Alignment of) Human Enolase β (ENOB) subunit with equivalent sequencesfrom other species (Mouse, Rat, Cow, Pig, Horse, Chicken, Cat, Dog,Rabbit and Sheep) depicting homology.

FIG. 14.

Alignment of Human Enolase γ (ENOG) subunit with equivalent sequencesfrom other species (Mouse, Rat, Cow, Pig, Horse, Chicken, Cat, Dog,Rabbit and Sheep) depicting homology.

FIG. 15. Human Enolase 241-260 citrullinated peptide induces CD4responses.

DR4 transgenic mice were immunized with human 241cit peptide. Ex vivoElispot was used to determine the IFNγ responses generated to the humanand mouse equivalent citrullinated (cit) peptides and the wild type (wt)sequences. IFNγ responses to citrullinated peptides in the presence ofMHC class II blocking antibody (L243), CD4 blocking antibody or CD8blocking antibody (A) or in CD4 depleted or enriched cell fractions (B)were assessed. IFNγ responses to shorter peptide sequences were alsotested (C). For all assays media only responses were used as a negativecontrol. For each experiment n=3. p values are shown.

FIG. 16. Characterisation of Responses in HHDII/DP4 Mice.

Transgenic DP4 mice were immunised with three doses of human (A-E) ormouse (F) Enolase 241cit peptide over three weeks. Splenocytes werecollected 21 days after the initial dose was administered. Ex vivo IFNyElispot were used to determine the response to Enolase 241 peptides.Responses to the human peptide were tested for avidity by peptidetitration (A), IL-10 secretion (B) and Granzyme B secretion (C). IFNγresponses in the presence of CD4 blocking antibody (D) and to shorterpeptide sequences were also tested (E). For all assays media onlyresponses were used as a negative control. For each experiment n=3. pvalues are shown.

FIG. 17. Human Enolase 241cit Peptide Provides an In Vivo SurvivalAdvantage in B16 Melanoma Anti-Tumour Studies in HHDII/DP4 TransgenicMice.

DP4 transgenic mice were challenged with B16DP4 tumour. Survival isshown for unimmunised control animals and animals immunised with Enolase241cit peptide four days after tumour implant. n=10. Statisticaldifferences between immunised and unimmunised control mice weredetermined by Mantel-Cox test, p values are shown, n=10.

FIG. 18. Enolase 241cit Peptide Provides a Survival Advantage Pan02(Pancreatic) Tumour Model.

DR4 transgenic mice were challenged with the Pan02 pancreatic carcinomaline expressing constitutive DR4. Four days after tumour challenge micewere immunised with human Enolase 241cit peptide and survival monitored.Statistical differences between immunised and unimmunised control micewere determined by Mantel-Cox test, p values are shown, n=10.

FIG. 19. Response can be Induced to Enolase 241cit Peptide inCombination with a Variety of Adjuvants.

DR4 (A& B) or HHDII/DP4 (C & D) transgenic mice were immunised withhuman Enolase 241cit in the presence of adjuvant CpG/MPLA (6 μg each),IFA, Poly I:C (10 μg) or Imiquimod (25 μg). IFNγ (A & C) and IL-10 (B &D) responses were determined by ex vivo Elispot. For these studies n=3.

FIG. 20. Enolase 241cit Specific Responses Reactivated from NormalDonors Produce Predominantly Th1 Cytokines and are CD4 Mediated.

PBMCs from donors 1 and 4 were cultured with human enolase 241cit or wtpeptide and IFNg release measured in IFNγ elispot at day 13 (A). IFNγresponses were analysed by intracellular cytokine staining incombination with CD4 and CD8 markers (B). Supernatant from PBMC culturesof donors 1, 4 and 7 were analysed for cytokine levels at days 2, 5 and12 by luminex assay (C).

FIG. 21. Eno 21cit Peptide Induces CD4 Responses in Mice.

C57Bl/6 mice were immunized with human enolase 21cit peptide. Ex vivoElispot was used to determine the IFNγ responses generated to the humancitrullinated (cit) peptide and the wild type (wt) sequences (A). IFNγresponses to citrullinated peptides in the presence of CD4 blockingantibody or CD8 blocking antibody (B) were assessed. For all assaysmedia only responses were used as a negative control. For eachexperiment n=3. p values are shown.

FIG. 22. Eno 11cit Peptide Induces CD4 Responses in Mice.

C57Bl/6 mice were immunized with human enolase 11cit peptide. Ex vivoElispot was used to determine the IFNγ responses generated to the humanand mouse citrullinated (cit) peptides and the human wild type (wt)sequences (A). IL-10 responses to the human cit peptide were assessed(B). IFNγ responses to human citrullinated peptide in the presence ofCD4 blocking antibody or CD8 blocking antibody (C) were assessed. Forall assays media only responses were used as a negative control. Foreach experiment n=3. p values are shown.

Methods

2.1. Commercial mAbs

Anti-HLA-DR antibody (clone L243) was purified from HB-55 hybridomacells (ATCC, USA) culture supernatant by sepharose protein G affinitychromatography. The antibody Rabbit monoclonal [EPR10864 (B)] to ENO1was used. Anti-mouse CD4 (clone GK1.5) and anti-mouse CD8 (clone 2.43)were purchased from BioXcell, USA.

2.2. Cell Lines

The murine melanoma B16F1 and murine Lewis lung carcinoma LLC/2 celllines were obtained from the ATCC. The murine Pan02 cell line wasobtained from the National Cancer Institute tumour repository. The B16F1cell line is cultured in RPMI medium 1640 (GIBCO/BRL) and LLC/2 andPan02 in DMEM. Both are supplemented with 10% FCS, L-glutamine (2 mM)and sodium bicarbonate buffered unless otherwise stated.

2.3. Immunogens

2.3.1. Peptides

Peptides>90% purity were synthesized by Genscript (New Jersey, USA).Stored lyophilized in 0.2 mg aliquots at −80° C. On day of use they werereconstituted to the appropriate concentration in PBS.

2.4. Plasmids

The mammalian expression vector pCMVSPORT6 encoding murine alpha Enolase(ENO-1) full length cDNA (IMAGE ID 5376359) was obtained from SourceBioscience.

To construct the plasmid pVITRO2 Human HLA-DP4, the nucleotide sequenceencoding the full length human HLA-DPA*0103 alpha chain flanked byFspI/EcoRI and the HLA-DPB*0401 beta chain flanked by BamHI/Sallrestriction sites were synthesized. Following sequence confirmation, theHLA-DPA*0103 chain was cloned into the FspI/EcoRI mcs2 of the vectorpVITRO2-hygro-mcs (Invivogen). The HLA-DPB*0401 chain was subsequentlyinserted into the BamHI/Sall mcs1 of the mammalian expression vectoralongside the alpha HLA-DPA*0103 chain present within mcs2.

To generate the HHDII plasmid, cDNA was synthesized from total RNAisolated from EL4-HHD cells. This was used as a template to amplify HHDusing the forward and reverse primers and sub cloned into pCR2.1. TheHHD chain, comprising of a human HLA-A2 leader sequence, the humanβ2-microglobulin (132M) molecule covalently linked via a glycine serinelinker to the α 1 and 2 domains of human HLA-0201 MHC class 1 moleculeand the α3, transmembrane and cytoplasmic domains of the murine H-2Dbclass 1 molecule, was then inserted into the EcoRV/HindIII sites of themammalian expression vector pCDNA3.1 obtained from Invitrogen.

To generate the plasmid pVitro 2 Chimeric HLA-DR401 cDNA was generatedfrom mRNA isolated from the splenocytes of transgenic HLA-DR4 mice. Thiswas used as a template to amplify the chimeric alpha and beta chainsseparately using forward and reverse primers that incorporated aFspI/EcoRI and BamHI/Sall sites respectively. On sequence confirmationfull length chimeric alpha chain comprising of murine H2-Ea with humanHLA-DRA alpha 1 domain was ligated into the FspI/EcoRI mcs2 of thevector pVITRO2-hygro-mcs (Invivogen). The beta chain comprising ofmurine H2-Eb with human DRB1*0401 Beta 1 domain was then inserted intothe BamHI/Sall mcs1 of the vector alongside the chimeric alpha chain.

To construct the IFNγ inducible plasmid pDCGAS chimeric HLA-DR401, thechimeric alpha and beta chains, were cloned into the pDCOrig vectordescribed elsewhere (Metheringham et al., 2009) in replacement of theheavy and light chain. The IFNγ inducible promoter consisting of a TATAbox and the GAS (IFNγ activated sequence) direct repeat enhancer elementwas amplified by PCR utilizing the vector pGAS-Luc (Agilent) as atemplate. The CMV promoter within each cassette was excised and replacedwith the IFNγ inducible promoter driving expression of the HLA-DR401chains within the pDCOrig vector backbone. Endotoxin free plasmid DNAwas generated using the endofree Qiagen maxiprep kit (Qiagen, Crawley)

2.5. Transfection

LLC2 cells were transfected using the Lipofectamine transfection reagent(Invitrogen) with 4 μg of the plasmid pVitro 2 Chimeric HLA-DR401 thatencodes both full length chimeric alpha and beta chains according to themanufacturer's instructions. The B16F1 cell line previously knocked outfor murine MHC class II by Zinc finger Technology (Sigma Aldrich) wastransfected with either the pDC GAS chimeric HLA-DR401 or the pVitro 2chimeric HLA-DR401 plasmids where chimeric HLA-DR401 is under expressionof the IFNγ inducible promoter or the constitutive promoter that drivehigh level expression respectively. Transfected cells were selected bygrowth in the presence of Hygromycin B (300 μg/ml) or zeocin (300μg/ml). Lines were cloned by limiting dilution and expression wasconfirmed by flow cytometry using the anti-human HLA-DR PE-Cy7conjugated antibody (clone L243) from eBioscience. Cells transfectedwith the IFNγ inducible plasmid where incubated overnight in the absenceor presence of murine IFNγ (30 ng/ml, Gibco life technologies) prior tostaining with the antibody.

The B16F1 cell line previously knocked out for murine MHC class I and IIby Zinc finger Technology (Sigma Aldrich) was transfected using theLipofectamine transfection reagent (Invitrogen) with 4 μg of each of theplasmids pCDNA3 HHDII and pVITRO2 Human HLA-DP4 plasmids. Transfectedcells were selected by growth in the presence of G418 (500 μg/ml) andHygromycin B (300 μg/ml). Lines were cloned by limiting dilution andexpression was confirmed by flow cytometry using the anti-human beta 2microglobulin FITC and anti-human HLA-DR/DP/DQ (clone WR18) PEantibodies from Serotec and Abcam respectively.

2.6 Western Blotting

Cell lysates were prepared in RIPA buffer containing protease inhibitorcocktail (Sigma) and proteins separated on a 4-12% NuPAGE Bis-Tris gel(Invitrogen) followed by transfer onto PVDF membrane. The membrane wasblocked for 1 hour with 3% BSA then probed with antibodies tohuman/mouse ENO-1 (clone EPR10863(B), Abcam) 1 in 1000 and β actin(clone AC-15, Sigma) 1 in 15000. Proteins were visualised using thefluorescent secondary antibody IRDye 800RD against rabbit (for ENO-1)and IRDye 680RD secondary anti mouse (for β actin). Membranes wereimaged using a Licor Odyssey scanner.

2.7. Immunisations

2.7.1. Immunisation Protocol

C57BL/6 mice (Charles River, UK), HLA-DR4 mice (Taconic, USA), HHDII/DR1mice (Pasteur institute, France) and the HHD/HLA-DP4 transgenic strainof mouse as described in patent WO2013/017545 A1 (EMMA repository,France) were used, aged between 8 and 12 weeks, and cared for by thestaff at Nottingham Trent University. All work was carried out under aHome Office project licence. Peptides were dissolved in PBS to 1 mg/mland then emulsified (a series of dilutions) with different adjuvants:CpG and MPLA 6 μg/mouse of each (Invivogen, UK), Incomplete Freund's 50μl/mouse (Sigma, UK), poly I:C 10 μg/mouse (Invivogen, UK), Imiquimod 25μg/mouse (Invivogen, UK) and GMCSF 10 μg/mouse (Peprotech, UK). Peptides(25 μg/mouse) were injected subcutaneously at the base of the tail. DNA(1 μg/mouse) was coated onto 1.0 μm gold particles (BioRad, HemelHempstead, UK) using the manufacturer's instructions and administeredintradermally by genegun (BioRad). Homspera (10 nM/mouse)(PeptideSynthetics, UK) was injected intradermally with genegunimmunisation. Mice were immunized at either day 0 for peptideimmunisation or days, 0 and 7 for genegun immunisation, unless otherwisestated. Spleens were removed for analysis at day 14 for peptide and day20 for peptide or genegun immunisation unless stated otherwise.

For tumour challenge experiments mice were challenged with 2.5×10⁴ B16DR4 cells or 1×10⁶ LLC2/LLC2 DR4 cells, 2.5×10⁵ Pan02 DR4 cells inmatrigel or 4×10⁵ B16 HHDII DP4 cells subcutaneously on the right flank3 days prior to primary immunisation and then were immunised as above.Tumour growth was monitored at 3-4 days intervals and mice were humanelyeuthanized once tumour reached 0 mm in diameter.

2.8. Analysis of Immune Response

2.8.1. Ex Vivo Elispot Assay

Elispot assays were performed using murine IFNγ, IL-17 and IL-10 captureand detection reagents according to the manufacturer's instructions(Mabtech, Sweden). In brief, anti-IFNγ, IL-17 and IL-10 antibodies werecoated onto wells of 96-well Immobilin-P plate. Synthetic peptides (at avariety of concentrations) and 5×10⁵ per well splenocytes were added tothe wells of the plate in triplicate. Tumour target cells were addedwhere relevant at 5×10⁴/well in triplicate and plates incubated for 40hrs at 37° C. After incubation, captured IFNγ and IL-10 were detected bybiotinylated anti-IFNγ and IL-10 antibodies and developed with astreptavidin alkaline phosphatase and chromogenic substrate. Spots wereanalysed and counted using an automated plate reader (CellularTechnologies Ltd).

2.8.2 Luminex Multiplexed Assay

A three-step indirect procedure was used for the multiplexed Luminexassay (Invitrogen) for IgG antibodies to IL-10, IL-17, IFNγ, TNFα, IL-2& IL-4. Standard, control, and unknown sera were diluted 1:2 in 50%assay diluent buffer (Invitrogen) & 50% serum free RPMI. Serial standarddilutions were included in each assay. Each dilution of standard,control, and unknown sera was mixed with a set of coupled Luminexmicrospheres in 96-well filtration plates (Millipore Multiscreen;Millipore Corporation, Bedford, Mass.) and incubated for 2 hours at roomtemperature with shaking. Microspheres were collected by vacuumfiltration and washed with PBST. Biotinylated detector antibody wasadded to each well for 1 hour at room temperature with shaking.Microspheres were collected by vacuum filtration and washed with PBST.Streptavidin conjugated R-phycoerythrin- was added to each well.Following a 30 minute incubation and a wash step, microspheres wereresuspended in PBST, and read in a Biorad BioPlex Luminex analyzerequipped with an XY platform. Data acquisition and analysis performedwith Luminex software (BioPlex Systems)

2.8.3 Proliferation Assay (Thymidine)

PBMC were isolated from freshly drawn heparinised blood by Ficol-Hypaque(Sigma) gradient centrifugation. PBMC (1.5×10⁶ cells/well) werestimulated with single peptides (final concentration 10 μg/ml) in RPMIcontaining 5% pooled autologous human serum, 2 mM glutamine, 20 mM HEPESand Penicillin-streptomycin (1%) in a final volume of 2 ml. Stimulationwith purified protein derivative, PPD (final concentration 10 μg/ml)served as a positive control for the proliferative capacity of PBMC. Asa negative control PBMC were incubated with medium alone. The PBMC werecultured at 37′C in an atmosphere of 5% CO₂ for 4, 7 and 11 days. Toassess proliferation at these times points 100 μl in triplicate fromeach culture was aliquoted into a round bottom well of a 96 well plateand ³H-thymidine added (0.0185 MBq/well) and incubated at 37° C. for afurther 8 hours. The cultures were harvested onto unifilter plates andincorporation of ³H-thymidine was determined by β-scintillationcounting. The results were assessed by calculating the stimulation index(SI) as the ratio of the mean of counts per minute (cpm) ofepitope-stimulated to the mean of unstimulated cultures. Theproliferative assay was considered positive when SI>2.5.

2.8.4 Proliferation Assay (CFSE)

PBMC were isolated from freshly drawn heparinised blood by Ficol-Hypaque(Sigma) gradient centrifugation. PBMC (1.5×10⁶ cells/well) werestimulated with single peptides (final concentration 10 μg/ml) in RPMIcontaining 5% pooled autologous human serum, 2 mM glutamine, 20 mM HEPESand Penicillin-streptomycin (1%) in a final volume of 2 ml. As anegative control PBMC were incubated with medium alone. The PBMC werecultured at 37° C. in an atmosphere of 5% CO₂ for 7 and 10 days. Toassess proliferation at these times points cells were sampled andstained with surface marker CD4 and CD8 antibodies labelled with PE-Cy5and efluor 450 respectively. After staining cells were fixed andanalysed on a Milteny MACSQuant flow cytometer.

2.8.5 PBMC Culture and IFNγ Elispot

PBMC were isolated from freshly drawn heparinised blood by Ficol-Hypaque(Sigma) gradient centrifugation. PBMC (1.5×10⁶ cells/well) werestimulated with single peptides (final concentration 10 μg/ml) in RPMIcontaining 5% pooled autologous human serum, 2 mM glutamine, 20 mMHEPES, Penicillin-streptomycin (1%), 10 ng/ml recombinant human IL-15and 5 ng/ml recombinant human IL-7 in a final volume of 2 ml.Recombinant human IL-2 was added on day 3 at 20 IU/ml. On day 13 cellswere washed and added to human IFNγ elispot assay. Elispot assays wereperformed using human IFNγ capture and detection reagents according tothe manufacturer's instructions (Mabtech, Sweden). In brief, anti-IFNγantibody was coated onto wells of 96-well Immobilin-P plate. Syntheticpeptides (at 10 μg/ml) and 1×10⁵ per well PBMCs were added to the wellsof the plate in quadruplicate and plates incubated for 20 hrs at 37° C.After incubation, captured IFNγ was detected by biotinylated anti-IFNγantibody and developed with a streptavidin alkaline phosphatase andchromogenic substrate. Spots were analysed and counted using anautomated plate reader (Cellular Technologies Ltd).

2.8.6 Intracellular Cytokine Analysis

PBMC cultures were set up as detailed above. On day 14 PBMCs were washedand cultured with synthetic peptide (10 μg/ml) in the presence ofbrefeldin A for 20 hrs at 37° C. Cells were stained with cell surfacemarkers CD4 and CD8 using PE-Cy5 and efluor 450 labelled antibodiesrespectively. Cells were subsequently fixed and permeabilised andstained with IFNγ PE-Cy7 labelled antibody. After staining cells werefixed and analysed on Miltenyi MACSQuant flow cytometer.

2.9 Immunohistochemical Analysis

Normal and tumour tissue binding was by immunohistochemistry (IHC) asdescribed previously (Durrant et al., 2006). Immunohistochemicalstaining was performed on 4 μm sections using Novolink polymer detectionsystem (Leica Biosystems, RE7150-K). Briefly, slides were deparaffinisedwith xylene and rehydrated through three changes of alcohol, theantigen-retrieval was performed in citrate buffer (pH 6.0) for 20 minusing a microwave oven. Endogenous peroxidase activity was blocked byPeroxidase Block for 5 min. Slides were washed with TBS (pH 7.6),followed by the application of Protein Block for 5 min. Followinganother TBS wash, primary antibody, optimally diluted in Leica antibodydiluent (RE7133), was applied and incubated for 60 min. The anti-ENO1rabbit monoclonal EPR10864 (B) was used at 1/200. Slides were washedwith TBS followed by incubation with Post-Primary Block for 30 minfollowed by a TBS wash. Novolink polymer was applied for 30 min. DABworking solution was made up of 1:20 DAB chromogen in DAB substratebuffer was prepared and applied for 5 min. Slides were counterstainedwith Novolink haematoxylin for 6 min, and dehydrated.

The TMA slides were initially assessed by light microscope assessment ofstaining quality and specificity. Slides were then scanned intohigh-resolution digital images (0.45 μm/pixel) using a NanoZoomer slidescanner (Hamamtsu Photonics, Welwyn Garden City, UK) and accessed usinga web-based interface NDP viewer (Nanozoomer Digital Pathology). Theywere scored at 920 magnification using a minimum of 2400 high-resolutionscreen (91920 1080). Cases were scored without knowledge of the ENO1status and patient outcome and were scored by two people (MG and MM).Assessment of staining was based on a semi-quantitative approach using amodified histochemical score (H-score) taking the intensity of stainingand the percentage of stained cells into account. For the intensity, ascore index of 0, 1, 2, and 3 corresponding to negative, weak, moderate,and strong staining intensity was used, and the percentage of positivecells at each intensity was estimated subjectively. Statistical analysiswas performed using SPSS 13.0 (SPSS Inc, Chicago). Stratificationcut-points for the survival analysis were determined using X-Tilesoftware (Camp et al., 2004) and P values of <0.05 were consideredsignificant.

Patient Cohorts

The study populations include cohorts of consecutive series of 462archived colorectal cancer (Simpson et al., 2010) specimens (1994-2000;median follow up 42 months; censored December 2003; patients with lymphnode positive disease routinely received adjuvant chemotherapy with5-flurouracil/folinic acid), 350 ovarian cancer (Duncan et al., 2007)samples (1982-1997; median follow up 192 months: censored November 2005:patients with stage II to IV disease received standard adjuvantchemotherapy which in later years was platinum based), 142 gastriccancer (Abdel-Fatah et al., 2013) samples (2001-2006; median follow up66 months; censored January 2009; no chemotherapy) 68 pancreatic and 120billary/ampullary cancer (Storr et al., 2012) samples (1993-2010: median45 months; censored 2012; 25-46% of patients received adjuvantchemotherapy with 5-flurouracil/folinic acid and gemcitabine) 220non-small cell lung cancers (January 1996-July 2006: median follow up 36months censored May 2013; none of the patients received chemotherapyprior to surgery but 11 patients received radiotherapy and 9 patientsreceived at least 1 cycle of adjuvant chemotherapy post-surgery)obtained from patients undergoing elective surgical resection of ahistologically proven cancer at Nottingham or Derby UniversityHospitals. No cases were excluded unless the relevantclinico-pathological material/data were unavailable. This retrospectivestudy was based on a consecutive series of 902 patients with primaryinvasive breast carcinomas who were diagnosed from 1987 to 1998 andentered onto the Nottingham Tenovus Primary Breast Carcinoma series.This is a well characterised series of patients under the age of 71years (median 55 years) with long term follow up. All patients weretreated in a uniform way in a single institution and have beeninvestigated for a wide range of protein expression.

All patients received standard surgical treatment of either mastectomyor wide local excision with radiotherapy. Before 1988, patients did notreceive systemic adjuvant therapy. From 1988 onwards, patients wereselected for systemic adjuvant treatment on the basis of NPI score andhormone receptor status. Patents with a NPI<3.4 received no adjuvanttherapy; those with an adjuvant score higher than 3.4 received tamoxifenif they were estrogen receptor positive (±goserelin if premenopausal) orclassical cyclophosphamide, methotrexate and fluorouracil if they wereER negative and fit enough to tolerate chemotherapy. Survival data wasmaintained prospectively. Breast cancer specific survival (BCSS) wasdefined as the time (in years) from the date of the primary surgicaltreatment to the time of death from breast cancer. Survival was censoredif the patient was still alive, lost to follow up (n=73) or died fromother causes.

Example 1. Sequence Alignment and Homology of Enolases

In mammals there are four isoforms of the enolase enzyme, ENO1 (A); ENO2(B), ENO3 (G) and ENO4 which are encoded by four distinct genes. Theyare highly conserved and have a high degree of amino acid homology (FIG.1).

Example 2. CD4 Responses to Citrullinated Enolase

The human alpha-Enolase peptide sequence was broken down intooverlapping 20-mers. Any 20-mer containing an arginine was selected andthe arginine residues were replaced with citrulline (cit). The selected20mer peptides are summarised in Table 1.

TABLE 1 Enolase peptides utilised.-indicates mouse and human sequences are homologousaa that alter in the mouse sequence are highlighted in bold Enolasepeptide (aa co- Peptide sequences ordinates) Human peptideMouse homologue   1-20 MSILKIHA-CIT-EIFDSRGNPTV MSILRIHA-CIT-EIFDSRGNPTV  6-25 IHA-CIT-EIFDS-CIT-GNPTVEVDLF IHA-CIT-EIFDS-CIT-GNPTVEVDLY  21-40EVDLFTSKGLF-CIT-AAVPSGAS EVDLYTAKGLF-CIT-AAVPSGAS  26-45TSKGLF-CIT-AAVPSGASTGIYE TAKGLF-CIT-AAVPSGASTGIYE  36-55PSGASTGIYEALEL-CIT-DNDKT —  46-65 ALEL-CIT-DNDKT-CIT-YMGKGVSKAALEL-CIT-DNDKT-CIT- FMGKGVSQA  56-75 -CIT-YMGKGVSKAVEHINKTIAP-CIT-FMGKGVSQAVEHINKTIAP 121-140 AGAVEKGVPLY-CIT-HIADLAGN — 126-145KGVPLY-CIT-HIADLAGNSEVIL KGVPLY-CIT-HIADLAGNPEVIL 171-190LPVGAANF-CIT-EAM-CIT-IGAEVYH LPVGASSF-CIT-EAM-CIT- IGAEVYH 176-195ANF-CIT-EAM-CIT-IGAEVYHNLKNV SSF-CIT-EAM-CIT- IGAEVYHNLKNV 241-260VIGMDVAASEFF-CIT-SGKYDLD VIGMDVAASEFY-CIT-SGKYDLD 246-265VAASEFF-CIT-SGKYDLDFKSPD VAASEFY-CIT-SGKYDLDFKSPD 256-275KYDLDFKSPDDPS-CIT-YISPDQ KYDLDFKSPDDPS-CIT-YITPDQ 261-280FKSPDDPS-CIT-YISPDQLADLY FKSPDDPS-CIT-YITPDQLADLY 316-335VGDDLTVTNPK-CIT-IAKAVNEK VGDDLTVTNPK-CIT-IAKAASEK 321-340TVTNPK-CIT-IAKAVNEKSCNCL TVTNPK-CIT-IAKAASEKSCNCL 326-345K-CIT-IAKAVNEKSCNCLLLKVN K-CIT-IAKAASEKSCNCLLLKVN 361-380QANGWGVMVSH-CIT-SGETEDTF QSNGWGVMVSH-CIT-SGETEDTF 366-385GVMVSH-CIT-SGETEDTFIADLV — 391-410 GQIKTGAPC-CIT-SE-CIT-LAKYNQL —396-415 GAPC-CIT-SE-CIT-LAKYNQLL-CIT- GAPC-CIT-SE-CIT-LAKYNQIL-CIT- IEEIEE 401-420 SE-CIT-LAKYNQLL-CIT-IEEELGSK SE-CIT-LAKYNQIL-CIT-IEEELGSK406-425 KYNQLL-CIT-IEEELGSKAKFAG KYNQIL-CIT-IEEELGSKAKFAG 416-434ELGSKAKFAG-CIT-NF-CIT-NPLAK ELGSKAKFAG-CIT-SF-CIT-NPLAK

Screening of Enolase Peptide Responses

Screening was performed to identify potential citrullinated Enolaseepitopes in mice. Mice were immunised with pools of 4-6 humancitrullinated peptides. To reduce the effect of possible crossreactivity the peptides within each pool were chosen so that they didnot contain any overlapping amino acid sequences. Each pool wasadministered as a single immunisation containing 20 μg of each peptideand CpG/MPLA as an adjuvant. After 14 days the mice were culled and theimmune responses to each peptide within the immunising pool wereassessed by ex vivo Elispot (FIG. 2). In addition, the splenocytes werescreened against the murine equivalent sequences. We have previouslyshown that citrullinated peptides can induce responses in the transgenicDR4 mouse strain. Given that different mouse strains have different MHCrepertoires a number of strains were used for screening. Peptideresponses were assessed in C57BL/6 mice as well as transgenic strainsexpressing human DR4 or HHD/DR1 in a C57BL/6 background (see materialsand methods).

Significant IFNγ responses were detected to a number of differentpeptides. In the DR4 mice the pool containing the Enolase 241-260citrullinated peptide induced a significant response to human 241cit(p<0.05) and mouse 241cit (p<0.0001). No other peptides showedsignificant IFNγ responses in DR4 mice. In the HHD/DR1 mice, the poolwith Enolase peptide 126-145 induced a significant response to human126cit (p<0.05) but not mouse 126cit. The pool with Enolase peptide316-335 induced a significant response to human 316cit (p<0.05) but notmouse 316cit. The pool containing the peptide Enolase 1-20 did notinduced a significant response to human peptide but did induce aresponse to mouse 1cit (p<0.05). In the C57BL/6 mice, the poolcontaining the peptide Enolase 21-40 induced a significant response tohuman 21cit (p<0.05) but not mouse 21cit. The pool with Enolase peptide126-145 induced a significant response to mouse 126cit (p<0.05) but nothuman 126cit. The pool with Enolase peptide 261-280 induced asignificant response to human 261cit (p<0.05) but not mouse 261cit. Thissuggests that peptides 21-40, 126-145, 241-260 and 316-335 justifiedfurther investigation.

From the initial screen Enolase 241cit immunisation in DR4 mice inducedthe strongest immune response. Therefore, this peptide was investigatedfurther. DR4 mice were given a single immunisation with 25 μg of thehuman 241cit peptide and CpG/MPLA. Ex vivo elispot on splenocytes showeda significant IFNγ response to citrullinated peptides compared to mediacontrols for both the mouse (p=0.0008) and human (p=0.0124) sequence(FIG. 3A). The mouse sequence is actually the same sequence as aa241-260from ENO2 and ENO3 so is still a self-antigen. Interestingly, neitherthe human or mouse wild type (wt) sequence, where the arginine residueat position 253 has not been replaced with a citrulline, produced animmune response. This confirmed that Enolase 241cit induced a citrullinespecific IFNγ response in DR4 mice.

To determine the type of cytokine response generated by Enolase 241citpeptide ex vivo IL-10 was also assayed. No significant increase in IL-10production was observed in ELISpot assays in response to peptidestimulation (FIG. 3B).

Previously citrullinated peptide specific responses have been shown tobe CD4 mediated. To determine whether the response to 241cit is CD4dependent an Elispot assay was performed with a human MHC class IIblocking antibody (clone L243) (FIG. 3C). IFNγ response weresignificantly reduced by L243 in response to both human Enolase 241cit(p=0.0171) and mouse 241cit (p=0.0023). To further confirm that 241citspecific responses were CD4 mediated Elispot assay was performedincluding a murine CD4 or CD8 blocking antibody (clone GK1.5 or clone2.43 respectively) or using CD4 enriched or depleted cell fractions.Responses to human enolase 241cit were significantly reduced in thepresence of the CD4 blocking antibody but not the CD8 blocking antibody(FIG. 15A). Responses were also present in the CD4 enriched fraction butnot in the CD4 depleted fraction (FIG. 15B). Since Enolase 241-260 is along peptide we sought to determine the optimal 15mer sequence that theresponses recognize. Two 15mer peptides spanning the 241-260 sequencewere tested for responses with aa241-255 stimulating specific responsesbut no response to aa246-260 (FIG. 15C).

To confirm lower frequency responses seen in initial screens, DR4,C57Bl/6 and HHD/DR1 mice were given a single immunisation of the humancitrullinated Enolase peptides corresponding to the sequences atpositions 21-40, 126-145 and 316-335 (FIG. 4). Enolase 21cit induced alow level but significant response in DR4 mice to the mouse (p<0.01) butnot to the human sequence. In C57Bl/6 mice strong IFNγ responses wereobserved that respond to the citrullinated but not the wt peptide (FIG.21a ). These appear to be CD4 mediated as they are efficiently blockedby CD4 blocking antibody but not a CD8 blocking antibody (FIG. 21b ).Enolase 126cit induced a low level significant response to the humanpeptide in DR4 mice (p<0.05) but not in DR1/HHD mice. Enolase 316citinduced a moderate response to the human but not the mouse sequence inboth DR4 (p<0.01) and DR1/HHD (p<0.01) mice.

Enolase sequences were also subject to in silico analysis for peptidesequences with high binding affinity to human and murine MHC class IIusing the online IEDB prediction program. This suggested the aa11-25sequence to be strong for murine MHC class II (1-Ab) therefore thecitrullinated aa11-25 peptide was tested for responses in C57Bl/6 mice.Mice showed IFNγ responses to this citrullinated peptide that crossreacted with the equivalent sequence from the murine sequence withminimal reactivity to the wt peptide (FIG. 22a ). No IL-10 responseswere seen to the citrullinated enol 1 peptide (FIG. 22b ). The enolase11 cit specific IFNγ response was mediated by CD4 cells as blockingthese with a CD4 blocking antibody abrogated the response whereas use ofa CD8 blocking antibody had no effect on the response (FIG. 22c ).

To determine whether HLA-DP4 might also be able to present the Enolase241cit peptide transgenic DP4 mice were utilised. DP4 mice wereimmunised with three doses of either human or mouse Enolase 241citpeptide. IFNγ responses were determined by Elispot (FIG. 5). Miceimmunized with human Enolase 241cit peptides showed responses to bothHuman Enolase 241cit (p<0.0001) and mouse 241cit (p<0.0001) showedincreased IFNγ responses when compared to the wild type peptides (FIG.5A). These responses show an avidity of between 1 and 0.1 ug/ml peptide(FIG. 16A). Cells from mice immunized with the human 241-260cit peptideshow granzyme B release but no IL-10 in response to the cit peptide butnot the wt peptide (FIGS. 16B and C). Responses specific for the human241-260 cit peptide in the DP4 mice are also blocked by a CD4 blockingantibody but not CD8 blocking antibody and show cross reactivity to ashorter peptide spanning aa241-255 (FIGS. 16D and E). Immunisation ofDP4 transgenic mice with the murine Enolase 241-260 peptide also inducesresponses specific to the citrullinated peptide but not the wildtype(FIG. 16F).

Example 3: Cit Enolase Peptide Presented on Tumour Cells can be Targetedfor Tumour Therapy

We had already established by Western blotting that the melanoma B16F1and Lewis lung Carcinoma cell lines constitutively express Alpha Enolase(FIG. 6A). Next, the anti-tumour effect of Enolase 241cit peptideimmunisation was assessed in vivo. The effect of Enolase immunisation onthe growth of the mouse B16 melanoma cell line transfected withconstitutive human DR4 (B16DR4) was assessed. Mice were challenged withB16DR4, 3 days prior to immunisation with Enolase 241cit peptide.Enolase 241cit peptide immunised mice showed a significant survivaladvantaged over control mice (FIG. 6B). Unimmunised mice showed 15%survival after 45 days whereas Enolase 241cit immunised mice showed 50%survival (p=0.0001). The tumour volume (FIG. 6C) was also significantlylower in the Enolase 241cit immunised mice (median 0 mm³) compared tothe control group (median 49 mm³) at day 17 post tumour implant(p=0.0043).

Since responses to the 241-260cit epitope have also been demonstrated inDP4 mice, DP4 transgenic mice were challenged with the mouse B16melanoma line expressing constitutive human DP4 (B16DP4) andsubsequently immunized with Enolase 241cit peptide. Enolase 241citpeptide immunized mice showed a significant survival advantage(p=0.0058) over unimmunized mice with survival rates after 60 days of70% and 10% respectively (FIG. 17).

To determine whether survival is effected by the constitutive expressionof MHC class II in this tumour cell line, the anti-tumour effect wasassessed in B16 cells where the HLA-DR4 expression is IFNγ inducible(iDR4). Mice were challenged with B16iDR4 4 days prior to immunisationwith Enolase 241cit peptide (FIG. 6D). Survival was significantlyincreased in the Enolase 241cit immunised group (90%) compared to theunimmunised control animals (survival 0%) at day 42 (p<0.0001). Thetumour volume at day 17 post tumour implant (FIG. 6E) was alsosignificantly lower in the Enolase 241cit (median 0 mm³) compared tounimmunised control mice (median 65 mm³, p=0.0048). Given the highsurvival percentage in the immunised group these mice were rechallengedwith B16iDR4 at day 42 to see if memory had been established. Newuntreated mice were also challenged with tumour as a control group.Enolase immunised survivors showed a significant survival advantage onrechallenge compared to previously untreated control mice (FIG. 6F). 39days after the rechallenge survival in the Enolase 241cit immunisedgroup was 67% while all of the control group were dead by day 29(p=0.0112).

To determine whether this anti-tumour effect is specific to the B16DR4model, mice were next challenged with the Lewis lung carcinoma cell lineLLC2 (FIG. 7A). Preliminary studies suggested that a higher implant cellnumber was required to obtain consistent growth of the LLC2 cellscompared to B16 cells (data not shown). For this reason, in our handsthe LLC2 model is more aggressive than the B16 model. Mice werechallenged with parental LLC2 or LLC2 transfected to constitutivelyexpress DR4 (LLCDR4) four days before immunisation with Enolase 241citpeptide. Survival data shows that Enolase 241cit immunisation provided asurvival advantage against the LLCDR4 tumour (p=0.0142) with 40% of micesurviving today 58 compared to the control where all mice died by day48. However, in mice challenged with the parental LLC2 tumour whileEnolase 241cit immunised mice showed a small but significant increase insurvival time (p=0.0005). These results suggest that human DR4expression on tumour cells is important for tumour rejection in thismodel.

Enolase is also expressed by the pancreatic tumour line Pan02 (FIG. 6A).This line was engineered to constitutively express HLA-DR4 and DR4transgenic mice were challenged with tumour followed 4 days later byimmunization with Enolase 241cit peptide. Enolase 241cit peptideimmunized mice show significantly enhanced survival (p=0.0076) in thePan02 DR4 model compared to unimmunized control DR4 mice. 50% of miceshow survival at day 60 compared to none of the unimmunized mice (FIG.18).

Example 4. DNA Immunisation Results in Responses to CitrullinatedEnolase

As APCs can constitutively citrullinate epitopes it was possible that aDNA construct encoding Enolase may be citrullinated and stimulate aresponse. HLA-DR4 transgenic mice were therefore immunised with a DNAconstruct encoding mouse enolase. Stimulated T cells from these micewere screened in vitro for IFNγ, responses to both citrullinated anduncitrullinated enolase 241 peptide. FIG. 8A shows that mice onlyresponded to the citrullinated mouse peptide (mean: IFNγ 180/millionsplenocytes; p=0.0001). No II-10 response was observed (FIG. 8B).

Next, the anti-tumour effect of Enolase DNA immunisation was assessed invivo. Mice were challenged with B16DR4 four days prior to immunisationwith Enolase DNA. Enolase DNA immunised mice showed a significantsurvival advantaged over control mice (FIG. 8C). Unimmunised showed 15%survival after 45 days whereas Enolase DNA immunised mice showed 60%survival (p=0.0001). The tumour volume (FIG. 8D) was also significantlylower in the Enolase DNA immunised mice (median 20 mm³) compared to thecontrol group (median 150 mm³) at day 17 post tumour implant (p=0.0088).

Example 5. Determination of Whether CD4 Responses to Enolase PeptidesVary when Combined with Different Adjuvants and at Different Doses

Enolase 241cit peptide induces a strong IFNγ response when administeredas a single 25 ug dose with the adjuvant CpG/MPLA. The effect ofadjuvant and dose regime on the response generated was investigated.Mice were immunised with a single dose of Enolase 241cit peptide witheither CpG/MPLA or incomplete Freund's adjuvant (IFA) as the adjuvant.IFNγ responses to Enolase 241cit peptides were detected by Elispot whenCpG/MPLA (p=0.0028) was the adjuvant but no IFNγ response was seen whenIFA was used as an adjuvant (FIG. 9A). IL-10 responses were detected byElispot when Enolase 241cit peptide was administered with IFA (p<0.0001)but not CpG/MPLA (FIG. 9B). In addition to these adjuvants responses incombination with other TLR agonists Poly I:C (TLR3) and imiquimod (TLR7)were assessed in both DR4 and DP4 transgenic mice. The combination withpoly I:C induces IFNγ responses in both mouse strains but no IL-10 (FIG.19). Enolase 241cit peptide combined with imiquimod induces IFNγresponses in DP4 transgenic mice but not in DR4 transgenic mice (FIG.19). This suggests that the type of cytokine response generated byimmunisation with Enolase 241cit peptide can be strongly affected by theadjuvant selected.

Next, dose responses to immunisation with GM-CSF were assessed. Micewere given a single or three immunisations with either 25 μg or 5 μg ofEnolase 241cit peptide. IFNγ responses were assessed by Elispot (FIG.9C). Detectable responses could be observed after 1 or 3 doses with 25μg of peptide. Next mice were challenged with B16DR4 and then giventhree doses of 5 μg of Enolase in GM-CSF over three weeks, to determinewhether this is sufficient to induce an anti-tumour response (FIG. 9D).Enolase 241cit immunised mice had a significant survival advantage overcontrol mice (p=0.0045) with 70% of animals surviving at day 45 comparedto 0% in the control group by day 28.

Example 6. Enolase 241cit Memory Responses

The ability of different adjuvants to polarise the responses toimmunisation with Enolase 241cit peptide may suggest plasticity of theT-cell population involved. This may indicate a pre-existing or memoryresponse. Therefore, next the speed with which an Enolase cit responsedeveloped was determined. Mice were immunised with a single dose ofEnolase 241cit peptide in CpG/MPLA 2, 6 or 14 days before beingsacrificed. Ex vivo Elispots were used to determine the IFNγ responses(FIG. 10). Immunisation with the mouse version of the peptide induced anIFNγ response which could be detected 2 days later. There was nosignificant difference between the responses seen after 2, 6 or 14 days.Immunisation with the human Enolase 241cit peptide led to an IFNγresponse which was detectable after 6 days. Responses were significantlyincreased after 6 (p=0.0009) and 14 (p=0.0092) days when compared toresponses after 2 days. These results suggest that there may be apre-existing response to Enolase 241cit peptide which is specific to theendogenous murine peptide.

Example 7. Responses in Healthy Human Donors

Mouse response to Enolase 241cit peptide can also be detected as earlyas 2 days after immunisation. This raised the question of whether humanshave a pre-existing response to Enolase 241cit peptides which can bedetected. To investigate this PBMCs were isolated from 6 healthy donorsand cultured in the presence of Human Enolase peptides. Thymidineproliferation assays were performed on the cells after 4, 7 and 11 daysand the proliferation index for each was calculated (FIG. 11A). 5/6 ofthe donors showed proliferation to Enolase 241cit peptide on at leastone of the samples days. For example, Donor 1 showed a proliferativeresponse to Enolase 241cit at day 11 (mean 20.4) and day 7 (mean 28.6)but not at day 4 (mean 0.8). Responses to Enolase 241 wt wereconsistently low at day 11 (mean 0.9), 7 (mean 1.2) and 4 (mean 0.3). Incontrast Donor 2 showed only a low level response at day 11 (mean 2.7)and Donor 6 was a non-responder. For each donor HLA types weredetermined and are shown on the figure.

Donor 4 gave a high proliferation index at day 4 (mean 12.5) and day 7(mean 28) and day 11 (4.4). This donor was chosen for further analysis.Supernatants were taken from cells at each time point and cytokinelevels were analysed by Luminex. The response above the background levelof the media only control was calculated for each cytokine (FIG. 11B).IFNγ and IL-10 gave citrullinated peptide specific responses whichincreased over time. Some increase in IL-17, Granzyme B and TNFα levelswere seen in wild type stimulated cells however these responses werehigher in the citrullinated peptide stimulated samples.

Next, PBMCs from donor 4 were labelled with Carboxyfluoresceinsuccinimidyl ester (CFSE) prior to ex vivo culture in the presence ofpeptides. On day 7 and 10 cells were removed and stained with anti-CD8and anti-CD4 fluorochome conjugated antibodies and analysed by flowcytometry (FIG. 11C). Of the proliferating CFSE^(low) population between73-96% of cells were CD4+ and 0-2% were CD8+. Enolase 241cit peptideshowed increased proportions of CFSE^(low)CD4⁺ cells compared to Enolase241 wt peptide. At day 10, 15% of the Enolase 241cit lymphocytes areCFSE^(low)CD4⁺ whereas 1% of the Enolase 241 wt peptides areCFSE^(low)CD4⁺.

IFNγ responses have also been shown by IFNγ elispot assay in which PBMCscultured for 13 days in Enolase 241 cit or wt peptides were restimulatedwith citrullinated or wt Enolase 241 peptide and cytokine releasemeasured. FIG. 20A shows results of the IFNγ elispot assay on donors 1and 4. Cells from both donors show responses to the citrullinatedpeptide but not the wt peptide. Further analysis of these responses byintracellular cytokine staining reveals IFNγ responses to be CD4mediated. FIG. 20B shows intracellular cytokine staining on PBMCs fromdonor 4 cultured for 13 days in Enolase 241cit peptide and restimulatedwith either citrullinated or wt peptide. IFNγ positive CD4 cells areobserved upon stimulation with the citrullinated peptide (0.44%) but notthe wt peptide. Luminex data from cultures on 3 donors shows IFNγresponses to the citrullinated Enolase 241 peptide with minimal responseto the wt peptide and low level IL-10, TNFα or IL-17 responses (FIG.20C).

These results suggest that healthy humans are able to develop a CD4proliferative response to Enolase 241cit peptide which is citrullinespecific and capable of producing Th1 cytokines.

Example 8. Immunohistochemistry

Citrullination is carried out by PAD enzymes and in particular the PAD2and PAD4 enzymes. These require high levels of calcium and are usuallyactivated in dead or dying cells or cells undergoing autophagy. Healthycells should not express citrullinated proteins but tumours due toeither hypoxia or nutritional stress will activate autophagy andcitrullinated enolase. Colorectal, gastric, lung, breast and ovariantumours were therefore stained for expression of enolase.

Colorectal Tumours:

232 colorectal tumours were stained with an ENO-1 specific monoclonalantibody (Table 2). 28% of tumours failed to stain, 56% showed weakstaining (Hscore 1-100), 13% moderate staining (Hscore 101-200) and 3%showed strong staining (Hscore 201-300) were most cells stainedintensely.

TABLE 2 Immunohistochemical staining of Colorectal tumour array forEno-1 Negative Low Moderate High Total H-  0 1-100 101-200 201-300 SCOREcores 655 129 30 8 232 28% 56% 13% 3%

Gastric Tumours:

70 gastric tumours were stained with an ENO-1 specific monoclonalantibody (Table 3). 16% of tumours failed to stain, 62% showed weakstaining (Hscore 1-100), 19% moderate staining (Hscore 101-200) and 3%showed strong staining (Hscore 201-300) were most cells stainedintensely.

TABLE 3 Immunohistochemical staining of gastric tumour array for Eno-1Negative Low Moderate High Total H-  0 1-100 101-200 201-300 SCORE cores11 44 13 2 70 16% 62% 19% 3%

Non-Small Cell Lung Tumours:

223 non-small cell lung tumours were stained with an ENO-1 specificmonoclonal antibody (Table 4). 20% of tumours failed to stain, 59%showed weak staining (Hscore 1-100), 17% moderate staining (Hscore101-200) and 4% showed strong staining (Hscore 201-300) were most cellsstained intensely.

TABLE 4 Immunohistochemical staining of non-small cell lung tumourstumour array for Eno-1 Negative Low Moderate High Total H- 0 1-100101-200 201-300 SCORE cores 45 132 37 9 223 % 20  59 17 4

Ovarian Tumours:

223 ovarian tumours were stained with an ENO-1 specific monoclonalantibody (Table 5). 42% of tumours failed to stain, 51% showed weakstaining (Hscore 1-100), 2% moderate staining (Hscore 101-200) and 5%showed strong staining (Hscore 201-300) were most cells stainedintensely.

TABLE 5 Immunohistochemical staining of Ovarian tumour array for Eno-1Negative Low Moderate High Total H- 0 1-100 101-200 201-300 SCORE cores93 115 5 10 223 % 42  51 2  5

Breast Tumours:

858 breast tumours were stained with an ENO-1 specific monoclonalantibody (Table 6). 28% of tumours failed to stain, 19% showed weakstaining (Hscore 1-100), 36% moderate staining (Hscore 101-200) and 17%showed strong staining (Hscore 201-300) were most cells stainedintensely.

TABLE 6 Immunohistochemical staining of Breast tumour array for Eno-1Negative Low Moderate High Total H-  0 1-100 101-200 201-300 SCORE cores239 165 310 144 858 % 28% 19% 36% 17%

Oestrogen Receptor Negative Breast Tumours:

249 oestrogen receptor negative breast tumours were stained with anENO-1 specific monoclonal antibody (Table 7). 8% of tumours failed tostain, 14% showed weak staining (Hscore 1-100), 55% moderate staining(Hscore 101-200) and 23% showed strong staining (Hscore 201-300) weremost cells stained intensely.

TABLE 7 Immunohistochemical staining of Oestrogen receptor negativebreast tumour array for Eno-1 Negative Low Moderate High Total H-  01-100 101-200 201-300 SCORE cores 19 36 136 58 249 % 8% 14% 55% 23%

Example 9 Homology of Enolase Between Different Species

Enolases are highly conserved between, mouse, dog sheep, cows, horse,pig and humans (FIGS. 12-14). As the vaccine induces T cell responses inhumans and mice and anti-tumour responses in mice, it can be assumedsimilar responses will be seen in other species.

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1. A peptide comprising, consisting essentially of or consisting of: anamino acid sequence selected from: VIGMDVAASEFFcitSGKYDLD,VIGMDVAASEFYcitSGKYDLD, EVDLFTSKGLFcitAAVPSGAS, EVDLYTAKGLFcitAAVPSGAS,KGVPLYcitHIADLAGNSEVIL, KGVPLYcitHIADLAGNPEVIL, VGDDLTVTNPKcitIAKAVNEK,VGDDLTVTNPKcitIAKAASEK, IFDScitGNPTVEVDLF, or IFDScitGNPTVEVDLY

wherein “cit” represents citrulline, or iii) the amino acid sequence ofi), with the exception of 1, 2 or 3 amino acid substitutions, and/or 1,2 or 3 amino acid insertions, and/or 1, 2 or 3 amino acid deletions in anon-citrulline position.
 2. A nucleic acid encoding the peptide ofclaim
 1. 3. A binding moiety that binds the peptide of claim
 1. 4. Thebinding moiety of claim 3, which is an antibody.
 5. A pharmaceuticalcomposition comprising the polypeptide of claim 1 and/or the nucleicacid of claim 2 and/or the binding moiety of claim 3 or claim 4, incombination with a pharmaceutically acceptable carrier.
 6. The peptideof claim 1 and/or the nucleic acid of claim 2 and/or the binding moietyof claim 3 or claim 4, for use in medicine.
 7. The peptide and/ornucleic acid and/or binding moiety for use of claim 4, for use in thetreatment of cancer.
 8. The peptide and/or nucleic acid and/or bindingmoiety for use of claim 5, wherein the cancer is breast cancer includingoestrogen receptor negative breast cancer, colorectal cancer, gastriccancer, non-small cell lung cancer, ovarian cancer including endometrialcarcinoma, pancreatic cancer including pancreatic ductal adenocarcinoma,leukaemia, melanoma, head and neck cancer or lung cancer.
 9. The peptideand/or nucleic acid and/or binding moiety for use of claim 7 or claim 8,wherein the use is human or veterinary use.
 10. The nucleic acid for useof claim 7, 8 or 9, wherein the nucleic acid encodes enolase.